Over the past three decades, research supported by this grant has had a major impact on our understanding of the how Sinorhizobium meliloti invades nodules and establishes the chronic intracellular infection that underlies the symbiosis with its legume host. Our work has also identified common bacterial functions that are important for both symbiotic and pathogenic bacteria to interact with their respective eukaryotic hosts, revealed the missing enzyme in vitamin B12 biosynthesis, and discovered a previously unrecognized, extremely highly conserved RNase. The proposed research addresses critical problems concerning how host antimicrobial peptides modulate S. meliloti's cell cycle and physiology during symbiosis, how the master regulator CtrA acts to control S. meliloti cell cycle progression and terminal differentiation during symbiosis, and how the RNase YbeY exerts its multiple biological roles. Plant-encoded NCR (Nodule Cysteine Rich) antimicrobial peptides play key roles in the striking process in which the bacteria undergo rounds of endoreduplication and terminally differentiate into bacteroids. We will extend our recent work that has offered new molecular insights into how these peptides exert their effects by identifying the biological activities of representative symbiotic NCR and glycine-rich host peptides, investigating the roles of ExoS-ChvI and FeuP-FeuQ signaling pathways in S. meliloti's response to NCR peptides and survival within host plant cells, developing a DNA-based strategy for making nodule NCR and glycine-rich peptides, and continuing to investigate how BacA and other S. meliloti functions provide resistance to the antimicrobial activity of NCR peptides. We have recently gained major insights into how S. meliloti, which has a tripartite genome, controls its cell cycle in the free-living state. A comparison with the well-studied Caulobacter crescentus cell cycle has not only revealed conserved regulatory features common to other -proteobacteria, but also many intriguing differences. We will gain insights into how regulation of the S. meliloti cell cycle has been adapted for symbiosis by elucidating the mechanism by which NCR247 alters cell cycle regulation and blocks cell division, defining the direct transcriptional targets of S. meliloti cel cycle regulator CtrA, and analyzing the role of CtrA in regulating physiological processes relevant to symbiosis. Our characterization of a symbiotically defective S. meliloti mutant led us to discover a previously unidentified RNase, YbeY, which is present in almost all bacteria and plays crucial roles in rRNA processing, 70S ribosome quality control, and small RNA regulation. We will follow up on our recent results by assessing the role of YbeY in B. abortus pathogenesis, completing our investigation of the role of YbeY in the maturation of the 3' terminus of 16S rRNA, gaining additional insights into the mechanism of 70S ribosome quality control mediated by YbeY and RNase R, identifying YbeY's cellular RNA targets, and identifying YbeY inhibitors that could be lead compounds for a potential new class of antibiotics.